CYP2E1 metabolizes and activates many toxicologic important compounds. Toxicity of these agents is enhanced by ethanol, due to induction of CYP2E1. Moreover, CYP2E1 is induced under a variety of physiological and pathophysiological conditions such as diabetes, obesity and non-alcoholic steatohepatitis. Further understanding the biochemical and toxicological properties of CYP2E1 will be important in providing mechanistic information on the actions of CYP2E1 in intact cells and this is the major goal of this application. AIM 1- Adaptation to oxidant stimuli is critical for survival of cells exposed to oxidative stress. We found an up- regulation of important antioxidant genes by CYP2E1. Nrf2 is a key transcription factor regulating these antioxidant genes. We will evaluate Nrf2 levels and actions in vivo and in vitro after treatment of mice and rats with ethanol or inducers of CYP2E1 such as pyrazole and acetone and in our HepG2 cell models which express human CYP2E1. CYP2E1 null mice will be used to evaluate whether CYP2E1 plays a role in regulating Nrf2 levels and induction of antioxidant genes by ethanol. SiRNA-Nrf2 will be used to down-regulate Nrf2 and the consequences of this on CYP2E1- and ethanol-induced oxidant stress and toxicity determined. Nrf2 null mice will be treated with ethanol acutely and chronically to attempt to develop an oral alcohol model of liver injury. AIM 2- Damage to mitochondria is an early event in the loss of cellular viability when cells with high levels of CYP2E1 are exposed to ethanol and other prooxidants. We propose to further characterize the effects of CYP2E1 and the role of CYP2E1 in the effects of ethanol on mitochondrial function and intactness. We will evaluate the onset of the mitochondrial permeability transition (MPT) in HepG2 cells expressing CYP2E1 or in hepatocytes from pyrazole and ethanol-treated rats exposed to prooxidants. The specific role of CYP2E1 in onset of the MPT by ethanol and prooxidants will be determined using CYP2E1 null mice and inhibitors. CYP2E1 has recently been shown to be present in the mitochondria. We have developed a HepG2 cell line which expressed CYP2E1 only in the mitochondria. We propose to study the functional consequences associated with mitochondrial CYP2E1 when such cells are exposed to ethanol and to prooxidants. AIM 3- Possible interactions between LPS and CYP2E1 or between CYP2E1 and the Fas system have not been evaluated yet these are all independent risk factors involved in alcoholic liver disease. We will explore the susceptibility and possible synergistic effect of CYP2E1 overexpression to LPS or to Fas antibody hepatotoxicity and assess the involvement of CYP2E1 in the increase of hepatotoxicity of LPS or Fas antibody-induced liver injury following pyrazole or ethanol pretreatment in mice. These studies may provide an experimental model to better understand mechanisms of ethanol-induced liver damage. The role of oxidative and nitrosative stress and of Kupffer cells in the CYP2E1 potentiation of toxicity will be evaluated to provide mechanistic insights of the toxicity process. It is hoped that such studies in hepatocyte and HepG2 cell culture models, and in vivo control and knockout mouse models will help to define further the biochemical and toxicological actions of CYP2E1 and its role in alcohol- induced liver injury. [unreadable] [unreadable] [unreadable] [unreadable]